MECHANISM OF DRUG RESISTANCE -...

CHAPTER 5

MECHANISM OF DRUG RESISTANCE

Single Strand Conformation Polymorphism 97

Characterization of Mutations in Drug Target Genes 102

Novel Mechanism of Drug Resistance Involving

Efflux Protein/s 122

Discussion 125

Mechanism of Drug Resistance

Single Strand Conformation Polymorphism

The principle of PCR-SSCP is based on the fact that the two denatured

strands of DNA (in this case PCR-amplified) adopt stable

intramolecular conformations which may differ from the wild type

upon mutation. This causes a change in the electrophoretic mobility

of the strands.

Princi_p(e of SSCP

.r~ ~T'

N

T~ ~r'

97


We utilized SSCP to conduct a primary screening of the rpo B, kat G

and gyr A amplicons for the presence of mutations. The results of

SSCP analysis reveal that while most of the rifampicin resistant

strains did exhibit the expected mobility shifts correlating with point

mutations, a very large percentage of isolates resistant to isoniazid

and fluoroquinolone that had point mutations (revealed subsequently

after sequencing), however did not display altered electrophoretic

mobility. Some typical SSCP gel electrophoresis patterns

corresponding to the rpof3 (Figure 5.1), katG (Figure 5.2) and gyrA

amplicons (Figure 5.3) are presented. To rule out any false negative we

did sequencing for all the suspected loci for all the drug resistant

Mycobacterium tuberculosis patient isolates included in our study.

Our findings indicate that certain sequences possibly lack secondary

structures or such different structures may not show mobility

differences. In such cases the SSCP will not be useful for screening for

mutations. With the advent of automated sequencing large number of

samples can now be directly sequenced to identify mutations.

Therefore the use of SSCP can be restricted to situations where

automated sequencing is not available.

98

A

c 1 2 3 4 5 6 7 8 9 10 11

B

c 1 2 3 4 5 6 7 8

Figure 5.1: The SSCP gel pictures of rpoB amplicons. The purified amplicons were fractionated on composite gel (5% polyacrylamide, 2% agarose). The wild type amplicon is loaded in lanes marked C in panel A and B. Arrows indicate bands which are not present in the wild type amplicon.

99

A

c 1 2 3 4 5 6 7

B

12 3 4 56 7 8 9 10 11 12 13 14 c

Figure 5.2: The pictures of silver stained SSCP gels with katG amplicons. In panel A the mobility differences in different samples are marked by arrow heads. Lane C in both panels has wild type amplicon. The samples in panel B demonstrate no mobility differences indicating no mutations. This however was proved wrong by sequencing which showed mutations.

100

A

1 2 3 4 5 6 7 8 9 c

B

1 2 3 4 c 5 6 7 8 9 10 11 12 13

Figure 5.3: The hotspot region in the gyrA loci was amplified by PCR. The amplicons were purified and fractionated on a composite gel (5% polyacrylamide, 2% agarose gel). The numbered lanes in panel A and B have samples while the lane marked C has the wild type amplicon. The arrow heads show the mobility differences indicating mutations in these sequences. In panel B the samples with S95T polymorphism were resolved. There is little mobility difference compared to the wild type.

101


Characterization of Mutations in Drug Target Genes

A large number of the patients (75%) had previous treatment history

and were on antitubercular treatment at the time of collection of the

sputa. Most of these cases had been through varying degree of

antitubercular drug therapy during the past 20 months. Of the total

126 isolates analysed, 73% (92) were resistant to rifampicin or a

combination of rifampicin with one or more drugs. This is supportive

of rifampicin being a surrogate marker for MDR tuberculosis in India

(Siddiqi et al., 1998).

Mutations in the hotspot regions of various loci were characterized.

The results are summarized in the Table 5.1. Based on the drug

susceptibility profile for an isolate, the corresponding loci

(representing the drug target gene) ~ere amplified and sequenced. The

largest number of samples was obtained fr'om Delhi followed by

Chandigarh, Ahmedabad, Agra, Bangalore, Shimla and few samples

from J aipur and Chennai. Except Chennai and Bangalore all the cities

are located in North India. We could establish previous treatment

history in 94 cases out of the total 126 cases included in the history.

These cases probably represent acquired resistance cases as the

patients had at some time point been given the antitubercular drug

therapy.

A stretch of 30 amino acids at the center of the amplicon for the rpo .13

locus was studied. The amino acids 432 to 458 comprised the hotspot

region for mutations. For the sake of comparison we used the

corresponding E. coli numbering which is 507 to 533.

102

Table 5.1: A summary of the patient isolates of Mycobacterium tuberculosis. The drug resistance profile and the mutation/ s in the corresponding target gene loci are shown. These samples were collected from the different cities in North-India and these are mentioned for each isolate. A 'yes' in the treatment history column indicates that the patient had received some kind of antitubercular drug therapy in the past twenty months.

STRAIN GEOGRAPHIC TREATMENT DRUG . POLYMORPHISM ID LOCATION HISTORYa SUSCEPTIBILITYb rpo katG/inhA gyrA rpsL

ICC14 N. Delhi Yes RR, IR, OR D516V N35D,NAat S95T 2"ct locus

ICC19 N. Delhi Yes. RR, IR, OR L511L, S531L R463L S95T ICC23 N. Delhi Yes RR IR

' L511L, S531L NA

ICC98 N. Delhi No IR, OR R463L, S95T Inh -err

ICClOO N. Delhi Yes RR, IR, OR, SR S531L R463L S95T NM ICC101 N. Delhi Yes RR OR

' S531L S95T

ICC102 N. Delhi Yes RR IR OR ' '

S531L NM S95T ICC103 N. Delhi Yes RR IR OR

' ' L511L, N518T NM A90A, S95T

ICC104 N. Delhi Yes RR IR OR ' '

D516V ~30C, R463L S95T ICC105 N. Delhi Yes RR, IR K527N R463L ICC107 N. Delhi No RR OR SR

' ' N518T, R528P S95T NM

ICC109 N. Delhi Yes IR NM ICClll N. Delhi Yes RR, IR, SR S531W Insertion 185C K121K ICC114 N. Delhi Yes IR R463L ICC115 N. Delhi Yes RR, IR S531W Insertion 98A,

R463L ICC123 N. Delhi Yes RR, IR, OR R528P NA S95T ICC124 N. Delhi Yes RR, IR H526Y R463L ICC125 N. Delhi Yes RR L511L, S531L ICC128 N. Delhi Yes RR, OR, SR H526Y, R528H S95T K121K

... Table 5.1 contd. ICC129 N. Delhi Yes RR IR SR

' ' R528P R463L K121K

ICC147 N. Delhi No RR IR OR ' '

S531W ~109G, R463L S95T ICC203 N. Delhi Yes RR D516V ICC204 N. Delhi Yes RR IR

' L521L, K527N R463L

ICC205 N. Delhi Yes RR, IR D516V NA ICC206 N. Delhi Yes RR, IR S531W NM ICC208 N. Delhi No RR D516V R463L ICC209 N. Delhi No RR D516V ICC210 N. Delhi Yes RR, OR L511V, N518T S95T ICC211 N. Delhi Yes IR, OR R463L S95T ICC212 N. Delhi No RR S531L ICC213 N. Delhi Yes RR, IR R528H, S531W R463L,

Inh -err ICC214 N. Delhi Yes IR R463L ICC215 N. Delhi Yes RR S531L ICC216 N. Delhi Yes RR, SR S531L K121K ICC217 N. Delhi Yes RR, IR, OR S531L R463L S95T ICC218 N. Delhi Yes RR IR OR

' ' S522Q R463L S95T

ICC219 N. Delhi Yes IR T12P, R463L ICC220 N. Delhi Yes RR, IR, OR, SR S531W R463L, D94G, S95T NM

Inh -T/A ICC221 N. Delhi No RR, OR L521L D94A, S95T ICC222 N. Delhi No OR S95T ICC223 N. Delhi No RR, OR H526Y S95T ICC225 N. Delhi Yes RR,OR S531L S95T ICC237 N. Delhi No RR, OR D516G S95T

... Table 5.1 contd. ICC239 N. Delhi Yes RR IR

' D516V R463L

ICC240 N. Delhi Yes RR, IR D516V R463L ICC242 N. Delhi Yes RR,OR L511V S95T ICC244 N. Delhi Yes RR, IR, OR S531L A61T, R463L S95T ICC246 N. Delhi Yes IR, OR Insertion 185C, S95T

R463L ICC275 N. Delhi Yes RR,OR H526Y S95T ICC277 N. Delhi Yes RR IR OR SR

' ' ' H526Y ~30C D94A, S95T K121K

ICC284 N. Delhi No OR NM ICC286 N. Delhi Yes RR IR

' D516G NM

ICC287 N. Delhi Yes RR, IR H526Y NM ICC325 N. Delhi Yes IR OR SR

' ' NM NM NM

ICC326 N. Delhi Yes RR, IR, SR H526L Insertion 98A, NM R463L

ICC327 N. Delhi Yes RR, IR, SR S509R R463L K121K ICC328 N. Delhi No OR NM ICC408 N. Delhi Yes RR IR OR SR

' ' ' Q510H, S531W R463L S95T K121K

ICC425 N. Delhi Yes RR IR '

S531L R463L F4 N. Delhi Yes RR OR

' H526Y D94G, S95T

F5 N. Delhi Yes RR OR ' S531L S95T

F7 N. Delhi Yes RR,OR S531L A90V, S95T F8 N. Delhi Yes RR, OR S531L A90V, S95T F9 N. Delhi Yes RR OR

' N518T S91P,S95T N31 N. Delhi Yes IR R463L N33 N. Delhi Yes IR R463L N34 N. Delhi Yes IR R463L

-0 VI

... Table 5 1 contd N35 N. Delhi Yes IR D73N,R463L N36 N. Delhi No IR R463L ICC32 Ahmedabad Yes RR, IR S531L NA ICC33 Ahmedabad Yes RR, IR, OR S509R R463L S95T ICC36 Ahmedabad Yes IR, OR R463L S95T ICC37 Ahmedabad Yes RR, IR D516G Insertion 98 A,

R463L ICC131 Ahmedabad Yes RR, IR H526Y, R528P R463L ICC132 Ahmedabad No OR S95T ICC133 Ahmedabad Yes· R\IR H526R ~30C, R463L ICC134 Ahmedabad No RR, OR S522Q S95T ICC136 Ahmedabad Yes RR, IR H526R Insertion 98 A,

R463L ICC137 Ahmedabad No OR S95T ICC138 Ahmedabad No OR S95T ICC226 Ahmedabad Yes RR, IR L511L, H526R ~109G, R463L ICC233 Ahmedabad Yes RR, IR D516V, H526Y ~30C, R463L ICC151 Chandigarh No OR S95T ICC154 Chandigarh No RR IR OR

' ' S531L ~30C,R463L S95T

ICC155 Chandigarh Yes IR ~30C, R463L ICC159 Chandigarh Yes RR, OR H526Y S95T ICC161 Chandigarh Yes RR,OR S522Q S95T ICC162 Chandigarh No RR,OR S522Q S95T ICC164 Chandigarh No OR S95T ICC165 Chandigarh No OR S95T ICC166 Chandigarh No OR S95T

-0 0'1

... Table 5.1 contd. ICC167 Chandigarh Yes IR, OR R463L S95T ICC168 Chandigarh Yes OR S95T ICC169 Chandigarh Yes RR, IR N518T R463L ICC170 Chandigarh No OR S95T ICC171 Chandigarh Yes RR IR

' S531L R463L

ICC172 Chandigarh Yes RR,OR S522Q S95T ICC173 Chandigarh No RR SR

' H526L K121K

ICC174 Chandigarh Yes OR S95T ICC175 Chandigarh No RR,OR H526Y S95T ICC247 Chandigarh No RR, OR D516V S95T ICC248 Chandigarh No RR,OR D516V S95T ICC249 Chandigarh No OR S95T ICC251 Chandigarh No OR NM ICC254 Chandigarh Yes RR S531L ICC255 Chandigarh No RR,OR N518T NM ICC256 Chandigarh Yes RR IR

' H526Y NM

ICC257 Chandigarh Yes RR,OR Q510H, L511L S95T ICC262 Chandigarh Yes RR, IR, OR D516V R463L S95T ICC95 Bangalore No OR S95T ICC96 Bangalore Yes RR,OR S531L S95T ICC399 Bangalore Yes RR, OR S531W NM ICC524 Bangalore Yes RR, IR S531L R463L ICC525 Bangalore Yes RR IR

' S531L R463L ICC143 Shimla No OR NM ICC144 Shimla Yes IR R463L ICC145 Shimla No OR NM

-s

... Table 5.1 contd. A3 Agra Yes RR, IR, SR S531L R463L NM A4 Agra Yes RR IR

' D516V R463L A9 Agra Yes RR IR

' S531L R463L All Agra Yes RR IR

' D516G TllA, R463L

A12 Agra Yes RR IR '

D516V N35D, R463L A13 Agra Yes RR IR

' D516V, N518T R463L

A14 Agra Yes RR, IR D516V R463L A15 Agra Yes RR IR

' S531L R463L

ICC332 Jaipur Yes RR IR '

H526R Insertion 185C, R463L

ICC337 Jaipur Yes RR, IR S531L R463L S95T ICC85 Chennai Yes RR IR OR

' ' H526R NA S95T

a: History of previous treatment b: RR-Rifampicin resistant; 1R-Isoniazid resistant; OR-Of1oxacin resistant; SR-Streptomycin resistant NA: No Amplification NM: No Mutation Inh: Mutation in the inhA ribosome binding site ~: Deletion at nucleotide position

-0 00


Many reported mutations as well as certain novel mutations were

identified. Codon 531 seemed to be the most vulnerable to mutations,

as most rifampicin resistant isolates had this mutation (Figure 5.4). Of

the 93 RIF resistant strains in our study, 28 had the mis-sense

mutation Ser531Leu and 8 had the substitution Ser531Trp. The next

most common mutations were the amino acid substitutions

Asp516Val/Gly (20 isolates) and His526Tyr/Leu/Arg (19 isolates). We

found two isolates with Gln510His changes. The figures 5.5a and 5.5b

show few of the typical electropherograms obtained by sequencing the

resistant isolates. While all these mutations have been reported

earlier, we also found mutations that have not been reported

previously. These include Ser509Arg (ICC33), Leu511Val (ICC242),

Asn518Thr (ICC107), Ser522Gln (ICC172), Lys527Asn (ICC105),

Arg528Pro (ICC129) and Arg528His (ICC213). Most of these mutations

were less frequent, comprising about 24% of the total mutations in the

94 isolates studied. Other mutations identified in our study were

silent mutations at the amino acids Leu511 and Leu521. Interestingly,

this mutation at position 511 never occurred alone and was only

present in isolates having more than one mutation at the rpo ..13 locus.

An important outcome of these studies is the direct correlation of

certain mutations with high MIC values. Table 5.2 lists the isolates,

their mutations and the corresponding MIC level till which they

remained resistant.

109

G G A G

G

• • • • 12?9 t Hl t t t t l t l t t t 1353 . ...ACC AGC CAG CTG AGC C,:AA TTC ATG GAC CAG AAC AAC CCG CTG TCG GGG TTG ACC CAC AAG CGC CGA CTG TCG. GCG

''T S Q L S Q F M D Q N N P L S G L T H K R R L S A

503 ~ ~ ~ ~ ~ ~ ~ . ~ ~ ~ 532

...... ...... 0

~ R H v T Q N p

• L

• G

y

• L H w

• R

Figure 5.4 : Summary of mutations at codons 508 to 532 in the rpoB gene. The wild type sequence and amino acids are shown in the middle frame. Nucleotide changes are marked with arrows in the top frame and the corresponding amino acid changes are denoted in the bottom frame. Changes marked in pink are novel mutations while silent mutations are marked in yellow .

...... ...... ......

, G C T T G T A. G G TC ,4 . . f14

' 190

1333 CAC --7 TAC H. 526 . Y

1333 CAC --7 GAC

H 526 D

G .,., G r c· .i\. ~· c· · It G r· · c L ~ . A j:l4i,:- ~Pi ' .:.

170

1349 TCG --7 TTG

S 531 L

Figure 5.5a: Some typical electropherograms showing the sequence of the rpoB hotspot region. The actual nucleotide and amino' a<?~d changes are marked below. The electropherograms are of the complement strand .

T C T GAT C CAT G .8. :1\ C T G C G AC ~0 . 2~ .

CAA1~6CAG Q 513 Q

GAC1~5 GAT D 516 D

C T GiTI; C C C .A. G T T C - ( ..• > 100 -

. 1291-92 . 1296 CAG ~ CTC CAA ~ CAG

Q 510 L Q 513 Q

C GACAGAC GAC GC TTGTGG .60 170 .

CGA 1~4CGT R . 529 R

GTTCTGGACCAT 210

~ 1

,1,

+ \ dt ~~ ~A II

r . ) '

J VJJf'v r:i . \/ ,1,, ;',;;!

1304 GAC ~ GTC

D 516 V

Figure S.Sb : Sothe typical electropherograms showing the sequence of the rpoB hotspot region. The actual nucleotide and amino acid change;\ are marked below. The electropherograms are of the complement strand.


Table 5.2: Specific mutations correlate with different minimum inhibitory concentration (MIC) values for rifampicin. Mis-sense mutations in the RpoB protein at amino acid positions 510, 511, 522, 526, 527, 528, 531 confer higher resistance (2:40~-tg/ml) levels than those at positions 509, 516 and 521 (_:510 1-lg/ml).

Level of

Rifampicin Amino Acid Strain Mutations Type

resistance change

(~-tg/ml)

ICC221 10 G1317A Novel L521L

ICC208 10 A1304T Reported D516V

ICC205

ICC37 10 A1304G Reported D516G

Gl317A Novel L521L ICC204 40

G1336T Novel K527N

ICC105 40 G1336T Novel K527N

ICC129 40 G1338C Novel R528P

C1331T Reported H526Y ICC131 40

G1338C Novel R528P

ICC123 64 G1338C Novel R528P

ICC100 64 C1349T Reported S531L

G1340A Novel R528H ICC213 64

C1349G Reported S531W

T1321C ICC218 64

C1322A Novel S522Q

G1287T Novel Q510H ICC257 64

C1288T Novel L5J1L

ICC275 64 C1333T Reported H526Y

ICC220 64 C1349G Reported S531W

C1331T Reported H526Y ICC128 128

G1338A Novel R528H

113


Mutations in codons 516 and 521 conferred low level resistance

(<40~g/ml) to rifampicin, whereas mutations in codons 510, 526, 527,

528 and 531 were seen to confer high levels of resistance (~64 ~g/ml).

The amino acids 526 to 531 appear to be very important in drug

target interactions, and mutations in them result in MIC levels in the

range of 64~g/ml and above. In a few cases (e.g. ICC204, 257 and

128) double mutations were found to have an additive effect on the

degree of resistance.

Insertion, deletion and substitution mutations were mapped in the

katG locus in 24 INH resistant isolates. In the present study we looked

for mutations in the 5' region (nt 3-239) and the mid-region (nt 1187-

1600) of the katG gene, corresponding to amino acid positions 2-77

and 395-533 respectively. The results are summarized in Figure 5.6. A

'C' nucleotide at position 30 was deleted in 6 of the isolates. This

deletion results in chain termination, thereby generating only a short

polypeptide of 26 amino acids. Another single G nucleotide deletion at

position 109 in 2 isolates was observed, which would result in the

production of a 45 amino acid long truncated polypeptide. Insertions

were also observed at nucleotide positions 98 (an A nucleotide) and

185 (a C nucleotide) in 4 and 3 isolates, respectively. Both these

insertions cause aberrant chain termination. Ala61Thr, Thr12Pro,

Thr11Ala, Asp73Asn and Asn35Asp mis-sense mutations were

observed in this locus in few of the isolates. These are novel

observations, as there are no reports of such mutations occurring in

INH resistant strains from other parts of the world.

114

...... ...... VI

+ + • t ~.

30nt 98nt 185nt

109nt

{/ II

1388nt

G .A. T C C T G G C .A. T 160

COG + CTG Arg 463 Leu

Figure 5.6: Summary of muta:tions in the katG gene. Deletions are indicated by orange lines superscripted with a ~ sign while insertions are depicted by black lihes superscripted with a+ sign. Substitutions are shown with blue lines. The electropherogram shows

t '- ~

the common mutation at the 463rd codon .

..... ..... 0\

A RBS

5----GAGAq GATAGG fTGTC----3

I ~ \ G T A/G

G.~ G ,AC G A TAG G T T G TC 130 1

GAG .AC GA iTAG G G T GTU 140 150''""'

G A G A T G .A. IT".~ G G T T G TC 130 140

C .... T

B c D

Figure 5.7: Panel A shows the known mutations in the putative RBS of inhA gene. The electropherogram showing the wild type sequence is shown in panel B. The panels C and D show the mutations observed in the patient isolates of Mycobacterium tuberculosis .


This locus could not be amplified in 6 of the isolates (ICC14, 23, 32,

85, 123, 205), indicating a partial deletion of the gene. A common

mutation in all these isolates was Arg463Leu (Figure 5.6). However,

this mutation has been shown to have no direct consequence for drug

resistance. The sequencing of this locus in isoniazid susceptible

isolates included in this study was done for confirmation. It was found

that the majority of the isolates carried this change. It has been

argued previously that this polymorphism in the katG locus might be

more important as a marker for evolution than resistance (Sreevatsan

et al, 1997)). Three INH resistant isolates carried mutations in the

ribosomal binding site (RBS) upstream of the inhA gene. While two

isolates showed a C to T transition, one had a T to A transversion

(Figure 5.7). These mutations have been reported previously by other

groups. The present understanding of these mutations is that they

probably confer resistance by a drug titration effect.

Sixty-eight ofloxacin resistant isolates were analyzed. The hotspot

region of the gyrA gene spanning codons 89 to 95 was sequenced to

identify mutations. Most of the isolates showed a single mutation

corresponding to the amino acid change Ser95Thr (Figure 5.8). The

second most common mutation, observed in four isolates, was

Asp94Gly I Ala (Figure 5.8, 5.9). Two isolates had Ala90Val

substitution while one had a silent mutation at this codon. Seven

isolates had double mutations, with the S95T change being common

to all. These mutations were present in MDR isolates that exhibited

high MIC levels to the drugs, including the frontline drugs used in

anti-tuberculosis therapy (ATI).

117

--00

T C

87 I i

c G

i CG

I I 96

2564 CAC GGC GAC GCG TCG ATC TAC GAC AGC CTG 2s9s

H G D I v

A I p

s I y D I

G A

s I T

L

Figure 5.8: Summary of mis-sense mutations in the gyrA locus. Nucleotide changes are indicated on top of the wild type sequence and the corresponding amino acid changes at the bottom. The most common mutation in this locus is Ser95Thr.


All the strains were also checked for mutations in the gyrB locus,

which is associated with low levels of resistance. However, we found

no mutations in the gyrB loci of these isolates. It has been argued that

the S95T mutation does not correlate with drug resistance (Sreevatsan

et al, 1997). It therefore appears that the isolates have acquired

resistance to ofloxacin via other mechanisms.

We tested 14 isolates resistant to streptomycin for mutations in the

rpsL and rrs loci. We found a novel silent mutation in eight strains at

amino acid position 121 in the rpsL locus (Figure 5.10) where the

codon AAA (Lys) was changed to AAG (Lys), but no mutations in the

rrs genes. To our knowledge there are no reports of this mutation. The

reported mutations at the rpsL locus are generally Leu43Arg,

Leu43Thr or Lys88Arg. We are still not clear about how this mutation

leads to the development of streptomycin resistance. The remaining

isolates probably acquired resistance by other means such as by

developing a permeability barrier or by producing drug-altering

enzymes.

A point to be kept in mind is that the majority of isolates included in

this study were from north India. Our data therefore, is inherently

biased towards drug resistant strains from this region and therefore

should not be seen as representing whole of India.

120

AAA Lys

--...... ~ AAG Lys

Figure 5.10: The electropherogram showing the novel silent mutation Lysl21Lys in the rpsL locus.

121


Novel Mechanism of Drug Resistance Involving Efflux Protein/ s

Rf-PCR with different sets of primer using RNA isolated from different ~

. drug resistant isolates was performed. These isolates were selected on

the basis of high level of MIC to the front line drugs such as

rifampicin, isoniazid and ofloxacin. Figure 5.11 depicts the amplicons

obtained by RT-PCR. The transcripts obtained from primers to genes

Rv783c, Rv1410c and Rv1819c demonstrated no appreciable

differences in their expression level under drug pressure. However, a

variance in the transcript level for Rv1258c obtained from isolate

ICC154 was observed.

The band intensity of 456 bp amplicon when divided by intensity of

the 245 bp band (from the constitutively expressed gene) provided a

means to compare the RNA transcript level of Rv1258c gene. The band

intensities were normalized in all the lanes and were subsequently

compared to each other. It is quite apparent that the levels of Rv1258c

RNA vary in presence or absence of different drugs from Figure 5.12.

Lane 1 and lane 4 represent the RNA from bacterium grown under

rifampicin and ofloxacin respectively. Lane 2 and 3 represent the

isolate grown in media containing isoniazid and no drug respectively.

The control H37Rv strain in lane 5 shows hardly any Rv1258c gene

RNA while the drug resistant isolate ICC154 shows an appreciable

increase in the corresponding RNA levels. There is a 10 fold increase

in the Rv1258c transcript level in isolate grown in the presence of

rifampicin and a six fold increase in the isolate when grown in

presence of ofloxacin. The drug target regions namely rpoB, katG and

gyrA in ICC 154 were amplified and sequenced.

122

A 1 2 3 4

+447 bp

+245 bp

c 1 2 3 4

B

D 1

1 2

2 3

3 4

4

456 bp

245 bp

•5o9 bp

Figure 5.11: RNA was extracted from M.tb. isolates grown in presence of low level of drugs-rifampicin (0.5~g/ml) , isoniazid (O.l~g/ml) and ofloxacin (O.l~g/ml). Out of the eight isolates only four grew in these conditions- ICC524, ICC337, ICC154 and ICC162. RT-PCR was done on RNA from these isolates using four set of primers corresponding to the genes Rv783c (Panel A), Rv1258c (Panel B), Rv1410c and Rv1819c (Panel D). A variance in transcript levels was observed in the case of Rv1258c (Panel B). Lane 2 in Panel D had very low amount of product possibly because of degradation of RNA. The bottom band (245 bp) in all the gels was the control RT-PCR amplicon.

123

M 1 2 3 4 5

500bp ___. .- 456bp

._ 245 bp Control

Figure 5.12: The RT-PCR products fractionated in a 2% agarose gel. Lane 5 represents the ~ontrol strain H37Rv. Lanes 1 to 4 show the amplicon generated from RNA extracted from ICC 154 when the isolate was grown in the presence of rifampicin, isoniazid, no drug and ofloxacin respectively. Lane M is the 100 bp DNA ladder.


There is deletion in the katG gene. The mutations in gyrA and rpoB

loci are generally associated with weak resistance. This correlates well

with the RT-PCR results. The high resistance to rifampicin and

ofloxacin could possibly be associated to overexpression of Rv1258c

product.

This gene was cloned and sequenced to look for any mutations. No

mutation in the gene isolated from this. particular isolate was

observed. To further understand the mechanism of up-regulation of

the transcript in the presence of the drugs the putative promoter

regions of this gene was examined. This gene has no recognized

promoter therefore 200 bp upstream of the translation start site of

this gene was sequenced and compared to the sequence of H3 7Rv.

Once again no changes in this region were seen.

Discussion

Resistance to drugs primarily arises due to mutations in genes coding

for drug target proteins (Ramaswamy and Musser, 1998). However,

this is not the exclusive mechanism employed by the pathogen to

evade killing by the drug. All mycobacteria, especially M. tb., have a

complex cell wall which blocks drug entry into the cell and thus

prevents the drug from accessing the cytoplasmic targets. A more

recent mechanism that is well documented in other systems is the

presence of membrane proteins that act as drug efflux pumps (Cole et

al, 1998; Lewis et al, 1994). The objective of this study was to identify

mutations in drug-target loci in Indian strains of Mycobacterium

tuberculosis and identify the different drug resistant genotypes. As in

125


all such studies, the aim was to generate information about the

markers associated with drug resistance, polymorphisms in the drug

target genes, association of level of resistance with particular

mutations, and for developing improved diagnostic and therapeutic

strategies to combat this menace. Rifampicin and isoniazid form the

backbone of the short course therapy for treating tuberculosis. These

drugs are the most effective, and resistance to them would necessitate

using drugs which are more toxic coupled with a longer treatment

period (Bloom et al, 1992).

The findings of mutations in the rpof3, katG and rpsL loci are similar to

those reported from other parts of the world, especially the common

mutations, which reflect a global pattern (Ramaswamy and Musser,

1998). The important feature of this study is the identification of novel

mutations, which assume significance while devising tools for the

diagnosis of MDR TB or designing alternate therapeutic strategies.

All mutations in the rpoB loci of the isolates fall in the 81 bp hot spot

region spanning amino acids 507 to 533. Resistance to rifampicin is a

relatively rare event and generally occurs only after resistance to other

components of short-course chemotherapy has already developed.

Therefore, rifampicin resistance is often regarded as an excellent

surrogate marker for MDR-TB (CDC, 1993; Hasnain et al, 1998). The

mutation frequency of codon 531 was similar to that reported earlier

by other groups (Kapur et al, 1994; Miller et al, 1994; Musser, 1995;

Ramaswamy and Musser, 1998; Siddiqi et al, 1998; Telenti et al,

1993a; Nachamkin et al, 1997; Telenti et al, 1997). Significantly, the

frequency of mutations (relative to other mutations) was higher at

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codon 516 and lower at codon 526 in Indian isolates compared to

those reported elsewhere (Ramaswamy and Musser, 1998). The novel

mutations reported in this broaden the range of known mutations at

this locus. When taken together, these mutations represent a

significant number of drug resistant isolates, a fact that needs to be

considered while designing tools for the detection of MDR TB. Many of

these mutations confer a high degree of drug resistance. A definite

correlation between MIC values and the type of mutation in many

isolates was observed. As reported by previous investigators

(Taniguchi et al, 1996) mutations at positions 528 and 531 are

important in developing high MIC levels. The present findings further

strengthen the belief that the degree of resistance to rifampicin

exhibited by an isolate is related to the type of mutation in the rpoB

locus.

In isoniazid resistant isolates significantly more deletion and

insertions than substitution mutations were found. It was observed

that almost all the isolates carried the Arg463Leu substitution.

However, this mutation was also found to be present in isolates that

were sensitive to INH. This is in concordance with a report from

Sreevatsan et al ( 1997) that argues that polymorphism at this residue

does not contribute to resistant per se but is an important marker for

evolutionary genetics. The insertions and deletions in the katG locus

invariably resulted in chain truncation and termination, leading to the

generation of dysfunctional polypeptides. Changes in the putative

ribosomal binding site of the inM gene in three isolates were also

seen. While the exact mechanism of how these mutations confer

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resistance to isoniazid is not clear, reports (Ramaswamy and Musser,

1998) indicate that they probably increase the levels of enoyl-ACP

reductase which in turn leads to resistance via a drug titration

mechanism.

Flouroquinolones comprise the secondary drug regimen in the

treatment of tuberculosis. Fluoroquinolones bind to DNA gyrase; the

g y r A and g y r B genes of M. tuberculosis have been cloned and

mutations in the quinolone-binding site have been mapped (Takiff et

al, 1994). Surprisingly, a large number of isolates resistant to

ofloxacin, was found. This could be in part due to the inaccurate

diagnosis of tuberculosis as a bacterial infection and fluoroquinolone

overuse in the population. The codons 89, 90, 91, 94 and 95 in the

gyrA gene have been shown to be polymorphic (Ramaswamy and

Musser, 1998; Xu et al, 1996). The most common mutation in

ofloxacin resistant isolates in this study was Ser95Thr. As reported

previously, there seems to be no direct role of this mutation in

developing drug resistance as it occurs in drug sensitive strains too

(Sreevatsan et al, 1997). It seems likely that ofloxacin resistance

possibly results due to mutations elsewhere in the gene or the

presence of drug efflux pumps.

Mutations in codons 43 and 88 of the rpsL gene result generally in

high levels of resistance to streptomycin, while mutations in the 530

loop or 915 regions of the rrs locus are associated with low levels of

resistance (Bottger, 1994). None of these mutations were seen in the

14 streptomycin resistant isolates included in our study. However, a

silent mutation at codon 121 that has not been reported by any other

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group was observed. Studies are underway to provide direct evidence

of the role of this mutation in conferring resistance to streptomycin.

Isolates that have no mutation in either gene may acquire resistant by

developing a permeability barrier to the drug.

Our study provides valuable data on different kinds of mutations

occurring at various target loci in Indian clinical isolates of M. tb. that

enhances our understanding of the molecular mechanisms of drug

resistance. The diversity of polymorphism exhibited at these loci by

the drug resistant strains indicates prevalence of a large number of

strains in this region. Additionally, these data will also assist in the

process of designing new molecular techniques for the diagnosis of

multi-drug resistant tuberculosis (Piatek et al, 2000; Wilson et al,

1999). Such methods promise faster detection rates compared to

methods solely based on the culturing of isolates.

Similar to other bacteria that demonstrate resistance to four or five

antibiotics, mycobacterium has also come up with novel means of

drug resistance. One of the mechanisms of resistance that has been

characterized in bacteria is the regulation and expression of genes

coding for drug efflux proteins. This study demonstrates, probably for

the first time, the association of similar efflux proteins to drug

resistance in Mycobacterium tuberculosis. Resistance in M. tb. has long

been associated with mutations in target genes. But studies indicate

other novel means of developing resistance.

In the clinical isolate ICC 154 included in this study we find a direct

correlation was found between RNA transcript levels of gene Rvl258c

and the different drugs included in the growth medium. The many fold

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increase in the transcript level when the isolate is grown under

rifampicin and ofloxacin point to the possibility of an operational

efflux pump. Studies carried out in M. smegmatis demonstrated the

efflux properties of the LFR protein (Liu et al, 1996; Sander et al,

2000) and increased levels of mRNA of pstB gene in ciprofloxacin

resistant strain (Banerjee et al, 1998). Both these genes are involved

in fluoroquinolone resistance. More recently two of the mdr-efflux

pump genes included in this have been cloned and expressed. Ainsa

et al ( 1998) have demonstrated that a recombinant plasmid containing

the Rv1258 gene when expressed in M. smegmatis confers low level

resistance to tetracycline (upto 4J..Lg/ml). This strengthens the data

discussed in the present study about this protein being a true drug

efflux pump. In a more recent study (Silva et al, 2001) the gene

Rv1410c homologue in M. bovis (termed P55) was expressed in M.

smegmatis. In this case also a weak tetracycline resistance was

observed. The question that needs to be answered is on the functional

regulation of these genes in the presence and absence of drugs. One

hypothesis is that these genes are part of operons where the other

units act as sensors for presence of drug and subsequently upregulate

the expression of the efflux pump genes.

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